A photodecarboxylase from Micractinium conductrix active on medium and short-chain fatty acids

doi:10.1016/S1872-2067(22)64173-1

Abstract

Abstract:

Hydrocarbons are essential base chemicals as energy carriers and starting materials for chemical manufacture. So-called fatty acid photodecarboxylases (FAPs) represent interesting catalysts for the conversion of natural fatty acids into hydrocarbons thereby giving access to alkanes from renewable feedstock. Today, however, only few FAPs are known. In the current study we report a new FAP from the marine organism Micractinium conductrix (McFAP). In contrast to currently known FAPs McFAP exhibits high catalytic activity towards short and medium fatty acids. Recombinant expression and basic biochemical characterisation of this new member of the FAP family is reported.

Key words: Photodecarboxylase, McFAP, Heterologous expression, Fatty acids, Hydrocarbon biofuel

Yunjian Ma, Xuanru Zhong, Bin Wu, Dongming Lan, Hao Zhang, Frank Hollmann, Yonghua Wang. A photodecarboxylase from Micractinium conductrix active on medium and short-chain fatty acids[J]. Chinese Journal of Catalysis, 2023, 44: 160-170.

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URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(22)64173-1

https://www.cjcatal.com/EN/Y2023/V44/I1/160

Figures/Tables 14

Scheme 1. The photodecarboxylase from Micractinium conductrix with a decarboxylation activity of medium and short-chain fatty acids.

Fig. 1. Phylogenetic tree analysis of photodecarboxylases.

Fig. 2. McFAP decarboxylation validation reaction. Reaction conditions: 0.25 g mL-1 McFAP@E. coli, 20 mg mL-1 CFE McFAP or 0.25 g mL-1 empty whole cells, 50 mmol L-1 fatty acid substrate, 30% (v/v) DMSO, pH 9 buffer (50 mmol L-1 Tris-HCl) were mixed and under gentle magnetic stirring (500 r min-1) at 30 °C in a total volume of 1 mL under the homemade photoenzymatic decarboxylation reaction setup (Fig. S1) for 12 h.

Fig. 3. SDS-PAGE analysis of the purified McFAP. Lane M: Protein Marker; Lane 1: whole cell protein; Lane 2: insoluble fraction; Lane 3: McFAP crude enzyme; Lane 4: purification passing sample; Lane 5: the protein sample eluted by buffer B.

Fig. 4. Sequence alignment results of CvFAP and McFAP.

Fig. 5. SDS-PAGE analysis of McFAP-S. Lane M: protein marker; Lane 1: protein sample of total bacteria after lysis; Lane 2: total bacteria are broken and centrifuged to supernatant protein samples; Lane 3: total bacteria are broken and centrifuged to precipitate protein samples; Lane 4: McFAP-S crude enzyme protein sample; Lane 5: pass through the liquid protein sample when purified by nickel column; Lane 6: Purified McFAP-S sample eluted by buffer B.

Table 1 Purfication of McFAP-S.

Sample	Total protein (mg)	Total enzyme activity (U)	Specific enzyme activity (U mg^-1)	Yield (%)
Crude enzyme solution	31800	2639	0.083	100
Affinity chromatography	836	334	0.399	13
After buffer change	595	159	0.268	6

Fig. 6. Comparison of efficiency of McFAP@E. coli and McFAP-S@E. coli catalyzing decarboxylation of different chain length fatty acids. Reaction conditions: 0.25 g mL-1 McFAP-S@E.coli or McFAP@E.coli, 50 mmol L-1 fatty acid substrate, 30% (v/v) DMSO, pH = 9 buffer (50 mmol L-1 Tris-HCl) were mixed and under gentle magnetic stirring (500 r min-1) at 30 °C in a total volume of 1 mL under the homemade photoenzymatic decarboxylation reaction setup (Fig. S1) for 1 h.

Fig. 7. Effect of temperature on McFAP-S activity (a), stability (b) and effect of pH on McFAP-S activity (c), stability (d). Reaction conditions: (a) Enzyme activity detection system: 20 μmol L?1 McFAP-S, 20 mmol L?1 n-octanoic acid, 15% (v/v) DMSO, 50 mmol L?1 Tris-HCl pH = 9 of 1 mL total system in a glass vials, 30 min, 500 r min?1, under the homemade photoenzymatic decarboxylation reaction setup (Fig. S1). (b) Enzyme activity detection system: 20 μmol L?1 McFAP-S, 20 mmol L?1 n-octanoic acid, 15% (v/v) DMSO, 50 mmol L?1 Tris-HCl pH = 9 of 1mL total system in a glass vials, 500 r min?1, under the homemade photoenzymatic decarboxylation reaction setup (Fig. S1). (c) For determination of pH optimum, the McFAP activity was measured at 30 °C using the following buffers: citrate-phosphate pH = 5 and 6, phosphate pH = 7, pyrophosphate pH = 8, Tris-HCl pH = 9 and 10 each at 50 mmol L?1 concentration. Enzyme activity detection system: 20 μmol L?1 McFAP-S, 20 mmol L?1 n-octanoic acid, 15% (v/v) DMSO, 50 mmol L?1 Tris-HCl pH = 9.0 of 1 mL total system in a glass vials, 30 min, 30 °C, 500 r min?1, under the homemade photoenzymatic decarboxylation reaction setup (Fig. S1). (d) McFAP-S pure enzyme solution and gradient pH buffer (pH = 6, 7, 8, 9, 10) were taken respectively and incubated for a certain time (0, 0.04, 0.13, 0.25, 0.5, 1, 2, 3, 4 and 5 d) under the condition of avoiding light at 4 °C. Enzyme activity detection system: 20 μmol L?1 McFAP-S, 20 m mol L?1 n-octanoic acid, 15% (v/v) DMSO, 50 mmol L?1 Tris-HCl pH 9 of 1mL total system in a glass vials, 30 °C, 500 r min?1, under the homemade photoenzymatic decarboxylation reaction setup (Fig. S1).

Fig. 8. Effect of illumination on the activity of McFAP-S (a) and residual activity of McFAP upon illumination in the presence of various fatty acids (b). (a) Pre-illumination conditions: blue light (400-520 nm), visible light i.e., sun light (380-780 nm), dark, red light (620-760 nm) were selected. McFAP-S pure enzyme solution (40 μmol L?1final) was incubated in the above light source at room temperature (25 °C) for a certain time (0, 10, 30 min, 1, 2 and 3 h). Sun light with C8:0 FA:10 mmol L?1final C8:0 FA. Calculate the residual enzyme activity of McFAP-S pure enzyme solution after incubation, reaction conditions: 24 μmol L?1 McFAP-S, 20 mmol L?1 n-octanoic acid, 15% (v/v) DMSO, 50 mmol L?1 Tris-HCl pH 9 of 1 mL total system in a glass vials, 30 min, 30 °C, 500 r min?1, under the homemade photoenzymatic decarboxylation reaction setup (Fig. S1). (b) Pre-illumination conditions: pre-illumination 2 h, 25 °C, under sun light (380-780 nm). Reaction conditions: 20 μmol L?1 McFAP-S, 20 mmol L?1 n-octanoic acid, 15% (v/v) DMSO, 50 mmol L?1 Tris-HCl pH 9 of 1 mL total system in a glass vials, 30 min, 30 °C, 500 r min?1, under the homemade photoenzymatic decarboxylation reaction setup (Fig. S1).

Fig. 9. Effect of enzyme concentration on the rate of the McFAP-S catalysed decarboxylation of n-octanoic acid (a) and time-conversion curve of purified McFAP-S catalyzing n-octanoic acid decarboxylation (b). General conditions: (a) 20 mmol L?1 n-octanoic acid, 15% (v/v) DMSO, 50 mmol L?1 Tris-HCl pH = 9.0 of 1 mL total system in a glass vials, 30 min, 30 °C, 500 r min?1, under the homemade photoenzymatic decarboxylation reaction setup (Fig. S1); (b) 60 μmol L?1 McFAP-S, 20 mmol L?1 n-octanoic acid, 15% (v/v) DMSO, 50 mmol L?1 Tris-HCl pH = 9 buffer of 1 mL total system in a glass vials, 30 °C, 500 r min?1, under the homemade photoenzymatic decarboxylation reaction setup (Fig. S1).

Fig. 10. Structural comparison of McFAP-S (cyan) and CvFAP (rose red) (a) and molecular docking of some carboxylic acids into the active sites of CvFAP (b) and McFAP-S (c). CvFAP: C10:0 (pink, 8.8 ?), C12:0 (rose red, 8.4 ?), C14:0 (blue, 7.5 ?), C16:0 (green, 4.8 ?); McFAP-S: C10:0 (pink, 2.6 ?), C12:0 (rose red, 6.3 ?), C14:0 (blue, 3.7?), C16:0 (green, 5.4 ?).

Fig. 11. Comparison of the catalytic performance of wt-McFAP-S and its triple mutant (S338A/L339I/T340S)-McFAP-S (a) and comparison of the catalytic performance of wt-McFAP-S and (?344-347)McFAP-S (b). Reaction conditions: (a) 40 μmol wt-McFAP-S or triple mutant (S338A/L339I/T340S)-McFAP-S, 50 mmol L?1 fatty acid substrate, 15% (v/v) DMSO, pH 9 buffer (50 mmol L?1 Tris-HCl) were mixed and under gentle magnetic stirring (500 rpm) at 30 °C in a total volume of 1 mL under the homemade photoenzymatic decarboxylation reaction setup (Fig. S1) for 1 h; (b) 40 μmol L?1 wt-McFAP-S or (?344-347)McFAP-S, 50 mmol L?1 fatty acid substrate, 15% (v/v) DMSO, pH = 9 buffer (50 mmol L?1 Tris-HCl) were mixed and under gentle magnetic stirring (500 r min?1) at 30 °C in a total volume of 1 mL under the homemade photoenzymatic decarboxylation reaction setup (Fig. S1) for 1 h.

Fig. 12. Structural comparison of wt-McFAP-S and (?344-347) McFAP-S. (a) structural of WT wt-McFAP-S. (b) structural of WT wt-McFAP-S. (c,d) the FFA substrate tunnel of wt-McFAP-S. (e,f) The 200ns MD simulation results. (g,h) MD trajectory analysis).

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